The present invention relates to a mixture comprising multiple native and/or denatured food allergens recognized by IgE antibodies of food-allergy patients, antibodies prepared by immunizing an animal with the mixture and a method for detecting the food allergens and allergy-inducing foods that involves using said antibodies. The invention provides safety to food allergy patients, since it is useful for understanding mechanisms of food-allergy onset, developing and verifying hypoallergenic foods, and detecting food allergens in foods, their raw materials and such food-manufacturing environments as food-manufacturing machines and processes.
Food allergy is a detrimental immunoreaction in susceptible individuals that is caused by ingestion of an allergy-inducing substance in a food (referred to as a food allergen in the following). The food allergy can cause dermatitis, asthma, digestive-tract obstacle, anaphylaxis shock, etc. Allergies are classified into types I to IV, depending on the mechanism of disease onset. Food allergy is primarily type-I allergy, in which IgE antibodies react with food allergens taken inside the body. The number of food-allergy patients has been increasing in recent years. Such a phenomenon causes serious problems in the fields of medical science and in the food industry.
To prevent such a hazard, it is necessary to provide information to the consumers through labeling. An FAO/WHO joint food standard committee (Codex Alimentarius Commision) has recognized the necessity of labeling of foods containing any of eight raw materials known as food allergens, and advised each of the member nations to implement the labeling system (June, 1999). In Japan, the labeling of 24 items was promulgated in consideration of the severity and frequency of food allergies (enforced since April, 2002). It should be noted that these regulations require the labeling of neither allergenic substance nor food allergen itself but that of food containing the allergenic substance and food allergen, namely allergy-inducing foods.
Allergy-inducing foods include eggs, milk, meat, fishes, the crustaceans and mollusks, cereals, legumes and nuts, fruits, vegetables, beer yeast, or gelatin. Similarly, ovoalbumin, ovomucoid, lysozyme, casein, beta-lactoglobulin, alpha-lactoalbumin, gluten, alpha-amylase inhibitor, etc. are known as allergy-inducing foods.
Furthermore, (1) regardless of present knowledge, it is conceivable that there are other many foods and ingredients (food allergens) that cause food allergy; (2) the allergy-inducing foods and ingredients (food allergens) are diverse and reactions to these allergens differ among food allergy patients; and (3) as the after-mentioned examples show, many known and unknown allergens exist even in a single allergy-inducing food. No conventional method, however, can easily detect so many allergy-inducing foods or food allergens.
Foods are prepared by such processes as heating, freezing, drying, salting, fermentation, enzymatic treatment, etc. (referred to as food-manufacturing processes in the following) to improve and stabilize their digestibility, shelf lives, taste, physical properties, etc. Although the food-manufacturing processes affect proteins and modify their molecular structures (e.g., denaturation of protein), it has seldom been discussed whether or not such food-manufacturing processes cause the formation of food allergens.
The present invention elucidated the following facts: (1) some heated food ingredients express allergenicity; (2) allergy-inducing components or epitopes of an individual allergy-inducing food may be altered, depending on whether the concerned food is heated or not. Namely, a) non-heated egg antigen, b) heated egg antigen prepared by heating the antigen in a), c) serum prepared by mixing the pooled serum from multiple food allergy patients (referred to as pooled patient serum) and a) (non-heated egg antigen-depleted serum), and d) serum prepared by mixing the pooled patient serum and b) heated egg antigen-depleted serum) were prepared. From the results of reactions between a) and c) or a) and d) and between b) and c) or b) and d), it was shown that the non-heated egg antigen-depleted serum could not immunologically react with the non-heated egg antigen but could react with the heated egg antigen: similarly, it was shown that the heated egg antigen-depleted serum could not immunologically react with the heated egg antigen but could react with the non-heated egg antigen. In summary, the food allergy patients carry IgE antibodies specific to both processed and non-processed foods, resulting in food allergy. So far, however, no simple method has been available to detect allergens in processed foods or their ingredients.
Methods screening for food allergens in chicken egg, peanut, casein, beta-lactoglobulin and gluten have been commercially available (Food and Development, Vol. 35, p 10-11). However, all or some of them possess one or more the following drawbacks: (1) The methods cannot always detect allergens that food allergy patients are sensitive to. In other words, they cannot always detect substances that are recognized by IgE antibodies of the patients; (2) The methods can detect known allergens, and a single allergen (single antigen) per method; (3) The methods using single antigen-detecting antibodies are not applicable to foods with inhibitory substances; (4) The methods using single antigen-detecting antibodies cannot exactly quantify the allergy-inducing foods as shown in the after-mentioned examples; (5) The methods using single antigen-detecting antibodies are not applicable to inspection of allergy-inducing foods containing no antigen (e.g., a method using a single antibody prepared by exposure to ovalbumin localized in egg white is not applicable to inspection of egg yolk, egg yolk mayonnaise, etc. and it is known that food allergy is caused by egg yolk); (6) The methods using single antigen-detecting antibodies are not applicable to inspection of the processed foods, because they cannot detect denatured or molecule-modified allergens; and (7) Monoclonal antibodies against native and denatured beta-lactoglobulin, ovoalubmin and alpha-casein have been reported (Allergy, vol. 50, p 309). Their usefulness may decrease, if the epitopes are removed or modified by the food-manufacturing processes, because the monoclonal antibodies only detect individual epitopes in a food-allergen molecule.
Although methods screening for food allergens using sera from patients who are allergic to rice (Japanese Patent Disclosure 2000-65820), egg and milk (Japanese Meat Science, vol. 39, p 166-169) have been reported, they are useful in hospitals but not in inspection organizations nor in food manufacturing factories, because the methods need a lot of sera from the patients. Although a food-allergen detection method depending on anaphylaxis reactions has been reported (FFI J., No. 180, p 77-82), it is not useful in most of the food-manufacturing factories because of complicated methodology and laboratory-animal husbandry. Although methods using flow system and enzyme-labeled antibodies and allergen sensors using microelectrodes have been developed (Japanese Food Industry, Vol. 42, p 53-56), many problems need to be solved before these methods can be put into practical use.